Project Summary Blockade of T cell costimulatory pathways represents a potent and highly specific method of preventing na[unreadable]ve anti-donor T cell responses following transplantation in mouse, monkey, and man. However, numerous studies have shown that the presence of donor-reactive memory T cells in the recipient poses a sometimes insurmountable barrier to long-term graft survival and tolerance induction. Despite these findings, it is increasingly well appreciated that a significant amount of heterogeneity exists within memory T cell populations, including with respect to their requirements for costimulatory signals during reactivation. We have discovered that high na[unreadable]ve donor-reactive T cell precursor frequency promotes the development of memory T cells that exhibit increased dependency on costimulatory signals for reactivation, and increased sensitivity to costimulation blockade following challenge with donor tissue. Therefore, in this application, we hypothesize that the conditions present during the priming phase of T cell activation lead to the differentiation of memory T cell populations with a higher or lower requirement for costimulation during secondary stimulation. We propose to rigorously dissect the differences in cell surface phenotype, effector function, and transcription factor expression in these donor-reactive memory T cells, with the goal of elucidating the factors critical for imparting upon some memory T cells their characteristic of costimulation independence. This issue has clinical relevance for the field of transplantation, in that if costimulation blockade is to successfully proceed in clinical application, we must understand the factors that make memory T cells more or less susceptible to costimulation blockade. The goal of this proposal is to investigate those parameters critical for programming the requirement for costimulation during the memory response to transplanted tissue.